Telomeres are terminal structural elements found at the end of chromosomes [Muller, The Collecting Net-Woods Hole, 13:181-195 (1939)] that protect natural double-stranded DNA ends from degradation, fusion, and recombination with chromosome-internal DNA [McClintock, Genetics, 26:234-282 (1941); Lundblad et al., Cell, 87:369-375 (1996)]. Telomeres are also thought to play a role in the architecture of the nucleus [Agard et al., Nature, 302:676-681 (1983); Rabl, Morphol. J., 10:214-330 (1885)], and to provide a solution to the end-replication problem that arises as a consequence of successive replication of linear DNA by DNA polymerases which would otherwise result with progressively shorter terminal sequences [Watson, Nature, 239:197-201 (1972)]. In tetrahymena, impaired telomere function leads to a defect in cytokinesis and to cell death [Yu et al., Nature, 344:126-132 (1990)]. Similarly, in yeast, loss of a single telomere results in cell cycle arrest and chromosome instability [Sandell and Zakian, Cell, 75:729-741 (1993)] and cells undergoing generalized telomere shortening eventually senesce [Lundblad and Szostak, Cell, 57:633-643 (1989); Singer and Gottschling, Science, 266:404-409 (1994)].
A ribonucleoprotein reverse transcriptase, telomerase, can elongate telomeres using an internal RNA component as template for the addition of the appropriate G-rich sequence to the 3' telomere termini [Greider and Blackburn, Cell, 43:405-413 (1985)]. This activity is thought to compensate for the inability of polymerases to replicate chromosome ends, but other mechanisms of telomere maintenance may operate as well [Pluta et al., Nature, 337:429-433 (1989)].
Telomeres contain a tandem array of repeat sequences, typically five to eight base pairs long, that are G-rich in the strand that extends to the end of the chromosome DNA. These repeat units appear to be both necessary and sufficient for telomere function [Lundblad and Szostak, Cell, 57:633-643 (1989); Szostak et al., Cell, 36:459-568 (1982)]. All telomeres of a single genome are composed of the same repeats and these sequences are highly conserved across species. For instance, Oxytricha chromosomes terminate in TTTTGGGG repeats [Klobutcher et al., Proc. Natl. Acad. Sci. USA, 78:3015-3019 (1981)], Tetrahymena utilizes an array of (TTGGGG).sub.n [Blackburn et al., J. Mol. Biol., 120:33-53 (1978)], and plant chromosomes carry the sequence (TTTAGGG).sub.n [Richards et al., Cell, 53:127-136 (1988)]. Telomeres of trypanosomes and all vertebrates, including mammals, contain the repeat sequence TTAGGG [Blackburn et al., Cell, 36:447-458 (1984); Brown, Nature, 338:774-776 (1986); Cross et al., Nature, 338:771-774 (1989); Moyzis et al., Proc. Natl. Acad. Sci. USA, 85:6622-6626 (1988); Van der Ploeg et al., Cell, 36:459-468 (1984)]. This 6 basepair sequence is repeated in long tandem arrays at the chromosome ends, which may be as long as 100 kb in the mouse, and varies from 2 to 30 kb in humans [de Lange, Telomere Dynamics and Genome Instability in Human Cancer, In Telomeres, Blackburn and Greider eds., Cold Spring Harbor Press; 265-295 (1995)].
During the development of human somatic tissue, telomeres undergo progressive shortening; in contrast, sperm telomeres increase with donor age [Broccoli et al., Proc. Natl. Acad. Sci. USA, 92:9082-9086 (1995); de Lange, Proc. Natl. Acad. Sci. USA, 91:2882-2885 (1994)]. Most if not all human somatic tissue chromosomes lose terminal TTAGGG repeats with each division, e.g., about 15-40 basepairs per year in the skin and blood. It is unclear what effect this diminution has since human telomeres are between 6-10 kb at birth. On the other hand, it is not yet known how many kilobases of TTAGGG repeats are necessary for optimal telomere function.
Primary human fibroblasts grown in culture lose about 50 basepairs of telomeric DNA per doubling (PD) before they stop dividing at a senescence stage [Allsopp et al., Proc. Natl. Acad. Sci. USA, 89:10114-10118 (1992)]. Importantly, there is an excellent correlation between the number of divisions that the cells go through and their initial telomere length. Indeed, it has been suggested that the correlation represents a molecular clock, which limits the potential of primary cells to replicate [Harley et al., Nature (London), 345:458-460 (1990); Harley et al., Exp. Gerontol, 27:375-382 (1992)]. Thus, immortalization of human somatic cells involves a mechanism to halt telomere shortening [Bodnar et al., Science, 279:349-352 (1998)].
Changes in telomeric dynamics also appear to play a role in the malignant transformation of human cells [Counter et al., EMBO J., 11:1921-1929 (1992); Counter et al., Proc. Natl. Acad. Sci. USA, 91:2900-2904 (1994); Kim et al., Science, 266:2011-2015 (1994)]. For example, telomeres of tumor cells are generally significantly shorter than those of the corresponding normal cells [de Lange et al., Mol. Cell Biol., 10:518-527 (1990)]. Telomerase activation appears to be an obligatory step in the immortalization of human cells [de Lange, Proc. Natl. Acad. Sci. USA, 91:2882-2885 (1994); Counter et al., EMBO J., 11:1921-1929 (1992); Counter et al., Proc. Natl. Acad. Sci., 91:2900-2904 (1994); Kim et al., Science, 266:2011-2015 (1994); Bodnar et al., Science, 279:349-352 (1998)].
Hanish et al. [Proc. Natl. Acad. Sci. USA, 91:8861-8865 (1994)] examined the requirements for the formation of human telomeres from TTAGGG seeds, and found that telomere formation was not correlated with the ability of human telomerase to elongate telomeric sequences in vitro, and did not appear to be a result of homologous recombination. Rather, the sequence dependence of telomere formation matched the in vitro binding requirements for TRF1, a telomeric TTAGGG repeat binding protein that is associated with human and mouse telomeres in interphase and in mitosis.
Indeed, several observations suggest the existence of regulatory mechanisms to control telomere length. Mammalian telomeres show a species-specific length setting [Kipling and Cooke, Nature, 347:400-402 (1990)] indicating a mechanism to control telomere length in the germline. Mammalian cells also have a mechanism to measure and regulate the length of individual telomeres. For example, in telomere seed experiments the final length of individual newly-formed telomeres matches the length of the host cell telomeres [Barnett et al., Nucl. Acids Res., 21:27-36 (1993); Hanish et al., Proc. Natl. Acad. Sci. USA, 91:8861-8865 (1994)]. Telomere length regulation is also apparent in several human cell lines, which maintain their telomeres at a stable length setting despite high levels of telomerase [Counter et al., EMBO J., 11:1921-1929 (1992)]. Thus, cells can monitor and modulate individual telomeres, a process that is likely to involve proteins bound to the TTAGGG repeats at chromosome ends.
Another process likely to be mediated by TTAGGG binding proteins is the protective cap function of telomeres. Telomeres are protected from the cellular surveillance systems that monitor DNA damage. Thus, cells can distinguish natural chromosome ends (telomeres) from double strand breaks (resulting from DNA damage).
The only known protein components of mammalian telomeres are the TRF proteins, duplex TTAGGG repeat binding factors that are localized at telomeres in interphase and metaphase chromosomes [Zhong et al., Mol. Cell. Biol., 13:4834-4943 (1992); Chong et al., Science, 270:1663-1667 (1995); Luderus et al., J. Cell Biol., 135:867-881 (1996); Broccoli et al., Hum. Mol. Genetics, 6:69-76 (1997); see Smith and de Lange, Trends in Genetics, 13:21-26 (1997) for review; Broccoli et al., Nature Gen., 17:231-235 (1997); Bilaud et al., Nature Gen., 17:236-239 (1997); van Steensel et al., Cell, 92:401-413 (1998)]. Thus far, only two human telomeric DNA binding proteins have been identified, TRF1 and TRF2 [U.S. Pat. No. 5,733,730, Issued Mar. 31, 1998, and U.S. patent application Ser. Nos. 08/938,052, filed Sep. 26, 1997, and 09/018,635 filed Feb. 4, 1998, all of which are whereby incorporated by reference in their entireties]. TRF1 was isolated as a double-stranded TTAGGG-repeat binding protein from HeLa cells [Chong et al., Science, 270:1663-1667 (1995)]. This factor contains three recognizable domains: an acidic N-terminal domain, a dimerization domain, and a C-terminal three helix bundle similar to the Myb and homeodomain DNA-binding folds [Bianchi et al., EMBO J., 16:1785-1794 (1997); Chong et al., Science, 270:1663-1667 (1995); reviewed in Konig and Rhodes, Cell, 85:125-136 (1996); Smith and de Lange, Trends in Genetics, 13:21-26 (1997)]. A second factor, TRF2, is related to TRF1 in its dimerization domain and the C-terminal Myb motif, but differs in that its N-terminus is basic rather than acidic [Bilaud et al., Nature Gen., 17:236-239 (1997); Broccoli et al., Nature Gen., 17:231-235 (1997)]. Despite their related dimerization domains, the proteins do not interact with each other [Broccoli, et al., Nature Gen., 17:231-235 (1997)], and probably exist predominantly as homodimers. Both proteins bind specifically to double-stranded TTAGGG repeats in vitro and are located at telomeres in vivo. The two TRFs are ubiquitously expressed and current evidence indicates that most human telomeres contain both factors bound simultaneously throughout the cell cycle [Broccoli et al., Nature Gen., 17:231-235 (1997); Chong et al., Science, 270:1663-1667 (1995); Smith and de Lange, Trends in Genetics, 13:21-26 (1997)]. Two other double-stranded telomeric-repeat binding proteins have been identified; Rap1p in S. cerevisia [Reviewed in Shore, Trends Gen., 10:408-412 (1994) and Taz1p in S. pombe [Cooper et al., Nature, 385:744-474 (1997)]. Both have Myb type DNA-binding domains [Cooper et al., Nature, 385:744-747 (1997); Konig et al., Cell, 85:125-136 (1996)]. In addition, Taz1p shows weak overall homology with TFR1 and shares its acidic nature [Cooper et al., Nature, 385:744-747 (1997)].
Recent studies have shown that TRF2 plays a key role in the protective activity of telomeres by inhibiting end-to-end fusions [van Steensel et al., Cell, 92:401-413 (1998)]. Previous studies had indicated that TRF1 plays a different role in telomere biology, functioning as a negative regulator of telomere length maintenance Ivan Steensel and de Lange, Nature, 385:740-743 (1997)]. Thus, long-term overexpression of TRF1 in a telomerase-positive tumor cell line resulted in progressive telomere shortening. Conversely, removal of TRF1 from the telomere (through expression of a dominant negative mutant) induced telomere elongation. In these experiments TRF1 did not detectably alter the activity of telomerase in cell extracts. Based on these observations it was proposed that TRF 1 negatively regulates telomerase at the level of individual telomeres; an increase in the amount of TRF1 at the telomere would create a negative signal for telomerase, whereas, a decrease would send a positive signal to telomerase [van Steensel and de Lange, Nature, 385:740-743 (1997)]. Interestingly, a similar mechanism of telomere length regulation exists in yeasts where it has been shown that Taz1p and Rap1p function as negative regulators of telomere length. As is the case for yeast telomere length regulation, the mechanism by which TRF1 controls telomere synthesis by telomerase is not fully understood [Conrad et al., Cell, 63:739-750 (1990); Cooper et al., Nature, 385:744-747 (1997); Lustig et al., Science, 250:549-553 (1990); Marcand et al., Science, 275:986-990 (1997); McEachern and Blackburn, Nature, 376:403-409 (1995)].
Indeed, telomere homeostasis involves a balance of lengthening and shortening activities. The telomerase catalytic subunit produces the lengthening activity, whereas other proteins including the telomere binding protein TRF1 are involved in establishing a telomere length equilibrium. Recently Bodnar et al. [Science 279:349-352 (1998)] have shown that extremely low levels of telomerase activity are insufficient to prevent telomere shortening; a result that is consistent with the observation that stem cells have low but detectable telomerase activity, yet continue to exhibit shortening of their telomeres throughout life.
Therefore, there is a need to isolate additional proteins, preferably enzymes involved in telomere homeostasis. Furthermore, there is a need to characterize such proteins. In addition, there is a need to design and develop drug screens to identify agents that modulate such proteins and thus can act as effectors on the important process of telomere length homeostasis.
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